Sequence diversity in pathogen antigens is an obstacle to development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family.
Every P. falciparum isolate has 50-60 diverse PfEMP1 variants, and the PfEMP1 repertoires of different isolates are largely non-overlapping [3,4,5,6]. PfEMP1 variants are expressed in a mutually exclusive fashion, and transcriptional switching from one var gene to another results in antigenic variation of P. falciparum infected erythrocytes [7]. PfEMP1 variants sampled from broad global parasite populations show essentially unlimited sequence diversity [5,8], making PfEMP1 an extremely challenging therapeutic target [9](see also: Pierce S K, Miller L H. J Immunol 2009; 182:5171-7).
Surface-reactive antibodies to PfEMP1 on live infected erythrocytes that occur after natural infections [10,11] or after immunization with recombinant PfEMP1 domains [11,12] are predominantly variant-specific, as expected for highly polymorphic parasite antigens. However, children living in endemic areas develop antibodies during the first few years of life that protect against life-threatening malaria [13] suggesting that strain-transcending antibody responses may occur, or that the parasites that cause severe malaria are of restricted antigenic types [14,15]. Antigenically-restricted subsets of parasite surface antigens that induce strain-transcending antibodies have not yet been identified.
In addition to their role in immunity and immune evasion, PfEMP1 variants are adhesion proteins that mediate interactions with a variety of human cell types and surface receptors [16,17]. Three major PfEMP1 families (A, B and C, based on conserved upstream sequence and genomic location) differ in their adhesive function [16]. Group B and C variants (approximately 40-50 variants per haploid parasite genome) bind to the endothelial protein and scavenger receptor CD36 [18,19]. In contrast, Group A variants (approximately 10 variants per haploid parasite genome) do not bind CD36 [18,19] but do mediate rosetting [11,12,20,21], an adhesion phenotype in which infected erythrocytes bind to uninfected erythrocytes [22]. Transcription of Group A var genes is linked to severe malaria in a variety of geographical settings [23,24,25,26] and laboratory experiments [27], whereas transcription of B and C var genes occurs in less virulent infections causing uncomplicated disease [23,24,25,26].
Rosetting is an important parasite virulence factor, associated with life-threatening malaria in African children [28,29,30,31,32] and high parasite burden in a primate malaria model [33]. Rosetting causes pathological obstruction to microvascular blood flow [34] and human erythrocyte polymorphisms that reduce the ability of P. falciparum to form rosettes confer substantial protection against severe malaria [35,36]. P. falciparum rosetting parasites can be divided into two distinct phenotypes: those that bind IgM natural antibodies (“non-immune” IgM) from normal human serum [37,38] and those that do not. Non-immune IgM binding is thought to strengthen the adhesion interactions between infected and uninfected erythrocytes in rosettes [37,39,40] and may also play a role in immune evasion by masking key epitopes [41]. Previous studies on PfEMP1 and rosetting have focussed on parasites with the non-IgM binding phenotype [11,12,20,21,42]. Detailed examination of IgM binding rosetting parasites has been neglected to date, despite the clinical importance of this phenotype, as rosetting parasites from clinically ill children are predominantly of the IgM binding type [38].